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ADME PK

BSEP, MRP2, MRP3 and MRP4 Inhibition

Understand the potential drug-bile salt interaction liabilities of your compound by using our BSEP, MRP2, MRP3 or MRP4 inhibition assays.

BSEP and MRP inhibition is in our portfolio of in vitro transporter services. Cyprotex deliver consistent, high quality BSEP and MRP inhibition data.

The effect of BSEP and MRP inhibition and their measurement in vitro

  • BSEP (bile salt export pump; ABCB11) is an ATP binding cassette (ABC) efflux transporter located on the canalicular membrane of hepatocytes1.
  • BSEP is the major transporter for the secretion of bile acids from hepatocytes into bile in humans1.
  • Bile secretory failure results in cholestasis. Bile secretory failure may be a result of;
    • Mutations in the gene encoding for BSEP which have been associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2) and benign recurrent intrahepatic cholestasis type 2 (BRIC-2)2.
    • Direct inhibition of BSEP by xenobiotics or drugs leading to acquired cholestasis and drug-induced liver injury (DILI)3.
  • Because of the link between BSEP inhibition and initiation of cholestatic DILI, the European Medicines Agency Guideline on the Investigation of Drug Interactions (2012) recommends in vitro screening of BSEP inhibition. If inhibition is observed then adequate biochemical monitoring including serum bile salts should be assessed during drug development4. Furthermore, the International Transporter Consortium recommends proactive evaluation and understanding of BSEP inhibition in drug discovery and development in order to aid internal decision making on the risk of DILI5,6.
  • MRP2 (multidrug resistance associated protein 2; ABCC2), MRP3 (ABCC3) and MRP4 (ABCC4) are ATP binding cassette (ABC) efflux transporters located on the canalicular membrane (MRP2) or sinusoidal membrane (MRP3, MRP4) of hepatocytes 5,6.
  • MRP2 transports conjugated bile acids from hepatocytes into bile, whereas MRP3 and MRP4 are able to transport bile acids from hepatocytes into blood5.
  • MRP3 and MRP4 efflux transporters are upregulated under cholestatic conditions suggesting they provide a protective role aginst bile acid-mediated hepatotoxicity by alleviating increases in intracellular bile acid concentrations, which may occur as a result of impaired biliary excretion due to inhibition of BSEP5,6,7.
  • Understanding whether a compound is able to inhibit MRP transporters may therefore provide useful additional information towards helping evaluate the risk of DILI.
  • Cyprotex offer BSEP, MRP2, MRP3 and MRP4 inhibition assays which investigate inhibition of the uptake of prototypical probe substrates (taurocholic acid for BSEP and estradiol 17β-D-glucuronide for MRPs) into inside-out membrane vesicles overexpressing the human ABC-transporter of interest.
Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk.

5Kenna JG et al., (2018) Clin Pharmacol Ther 104(5); 916-932

Protocol

BSEP, MRP2, MRP3 and MRP4 inhibition assay protocol

Test System Sf9 insect cell-derived or mammalian (HEK293) cell-derived inside-out membrane vesicles overexpressing a single transporter (BSEP, MRP2, MRP3 or MRP4) incubated in the presence of ATP and AMP (absence of ATP).
Probe Substrate [3H]-Taurocholic acid (BSEP)
[3H]-Estradiol 17β-glucuronide (MRPs)
Test Article Concentrations 6 concentrations plus 0 μM (triplicate wells) (final test compound concentrations dependent on customer requirements)
Time Point Dependent on transporter
Analysis Method Radiochemical detection using scintillation counting
Data Delivery IC50
Written report available on request
Related Services

P-gp
BCRP
Human SLC Transporters

Data

Data from Cyprotex's BSEP, MRP2, MRP3 and MRP4 inhibition assays

Figure 1
Mean human BSEP-mediated taurocholic acid transport in the presence of a range of concentrations of ketoconazole expressed as a percentage of vehicle control.

The results represent the mean ± standard deviation of 3-9 replicate wells (triplicate wells per incubation condition performed on three separate occasions).
Figure 2
Mean human MRP2-mediated estradiol 17ß-D-glucuronide transport in the presence of a range of concentrations of MK-571 expressed as a percentage of vehicle control.

The results represent the mean ± standard deviation of 3-9 replicate wells (triplicate wells per incubation condition performed on three separate occasions).
Figure 3
Mean human MRP3-mediated estradiol 17ß-D-glucuronide transport in the presence of a range of concentrations of terfenadine expressed as a percentage of vehicle control.

The results represent the mean ± standard deviation of 3-9 replicate wells (triplicate wells per incubation condition performed on three separate occasions).
Figure 4
Mean human MRP4-mediated estradiol 17ß-D-glucuronide transport in the presence of a range of concentrations of MK-571 expressed as a percentage of vehicle control. 

The results represent the mean ± standard deviation of 3-9 replicate wells (triplicate wells per incubation condition performed on three separate occasions).
 
TransporterSubstrateInhibitorMean IC50 ± Standard Deviation (µM)
BSEP Taurocholic acid Ketoconazole 8.78 ± 1.25
MRP2 Estradiol 17β-glucuronide MK-571 22.6 ± 6.38
MRP3 Estradiol 17β-glucuronide Terfenadine 33.5 ± 6.77
MK-571 56.8 ± 7.23
Fidaxomicin 1.06 ± 0.117
MRP4 Estradiol 17β-glucuronide MK-571 0.555 ± 0.238
Indomethacin 3.79 ± 0.342
Ibuprofen 42.0 ± 23.7
Table 1
Inhibition of human BSEP- and MRP-mediated transport of the prototypical substrates, taurocholic acid and estradiol 17β-glucuronide, respectively.

The incubation conditions for each of the species have been fully characterised for the chosen substrates, based on time linearity and uptake kinetics (Vmax and Km). The chosen substrate concentration is much lower than the determined Km, and as such IC50 equates to Ki (assuming competitive inhibition).

References

1 Wang L et al., (2002) The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II. J Clin Invest 110(7); 965-972
2 Dawson PA, Lan T, and Rao A. (2009) Bile acid transporters. Journal of Lipid Research 50(12); 2340-2357
3 Stieger B (2010) Role of the bile salt export pump, BSEP, in acquired forms of cholestasis. Drug Metab Rev 42(3); 437-445
4 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
5Kenna JG et al., (2018) Can bile salt export pump inhibition testing in drug discovery and development reduce liver injury risk? An International Transporter Consortium perspective. Clin Pharmacol Ther 104(5); 916-932
6 Zamek-Gliszczynski MJ et al., (2018) Transporters in drug development: 2018 ITC recommendations for transporters of emerging clinical importance. Clin Pharmacol Ther 104(5); 890-899
7 Morgan RE et al., (2013) A multifactorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development, Toxicol Sci 136; 216-241

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